Primate embryonic stem (ES) cells and the recently-described induced pluripotent cells (iPS) (collectively, “pluripotent cells”) can proliferate without limit and can differentiate into each of the three embryonic germ layers [1-3]. To facilitate self-renewal, primate (including human) pluripotent cells are typically co-cultured with mouse embryonic fibroblast (MEF) feeder cells, or cultured in MEF-conditioned medium (MEF-CM) on a Matrigel® extracellular matrix or in a chemically-defined medium. It is understood that iPS cells behave in culture essentially as do ESC. iPS cells and ESCs express one or more pluripotent cell-specific marker, such as OCT-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and Nanog. Subsequent references herein to embryonic stem cells, ES cells, human embryonic stem cells, hES cell and the like are intended to apply with equal force to iPS cells.
Cell microenvironment influences embryonic stem (ES) cell differentiation [4, 5]. For example, spontaneous differentiation of ES cell cultures occurs along seemingly random pathways during normal cell culture, especially as colony density and size increase [2, 6]. Typically, however, ES cell differentiation is stimulated either by co-culturing the cells with cells of particular lineages or by chemically or mechanically detaching the cells from their substrate to generate embryoid bodies (EBs) [2] that are cultured in suspension in the absence of MEFs or MEF-CM [7-11]. After several days, EBs in the suspension culture are plated to promote proliferation and further cell differentiation.
Interestingly, EBs in a single culture differentiate to distinct cell lineages. Subtle microenvironment differences in and around individual EBs are thought to affect differentiation of cells in EBs, which then further guide differentiation of other cells by cell-cell contact or by secretion of soluble differentiation factors [6]. One factor that may regulate lineage commitment is EB size [12]. For example, Ng et al. showed efficient generation of hematopoietic cells from “spin EBs” (i.e. EBs generated by centrifugation) having a uniform, yet large size, although the actual number of hES cells aggregating to form these EBs was not known [13]. Smaller spin EBs preferentially differentiated along other lineages. Unfortunately, the art lacks simple methods for producing EBs of consistent and desired size from ES cells.
One way to direct culture of some cell types, including 3T3 fibroblasts [14-17], capillary endothelial cells [18-20], mouse melanoma cells [17] and buffalo rat liver cells [17], is to constrain the cells within a patterned area on a two-dimensional (2-D) monolayer. Micron-scale patterns can be formed in self-assembled monolayers (SAMs) by micro-contact printing alkanethiols that spontaneously assemble via a linkage of a terminal sulfur group to sites on a gold substrate. The SAMs reach equilibrium within one to five hours [19].
Suitable alkanethiols typically contain an eleven to eighteen carbon chain and are capped with a functional group. Depending upon the nature of the functional group, SAMs can attract or repel extracellular matrix (ECM) proteins [16-24]. A common protein-repelling alkanethiol is poly-ethylene glycol (PEG)-terminated alkanethiol containing three to six ethylene glycol groups [16, 20]. Tri-ethylene glycol (EG3)-terminated alkanethiols resist protein and extracellular matrix adsorption for approximately eight days, but thereafter begin to break down under typical cell culture conditions [16]. In contrast, several alkanethiols, including methyl- and amine-terminated molecules, attract extracellular matrix proteins [19, 21, 22, 25-27].
Unfortunately, 2-D SAM monolayers are of limited utility for culturing primate ES cells because of the cell's growth nature. Unlike many cells, primate ES cells, including human ES cells, do not grow to confluence as monolayers and are not contact inhibited, but rather build upon themselves to form cell aggregates [28] that spread beyond the constrained areas of the 2-D monolayers. Likewise, initial efforts at 2-D micro-contact printing of Matrigel® on SAM surfaces indicated that this method was not suitable for long-term hES cell culture because of substrate instability and because growing colonies could span across unpatterned regions.
With few exceptions, current literature regarding patterned 2-D monolayers focuses primarily on cell attachment and replication to generate confluent monolayers in patterned regions, but does not investigate effects of three-dimensional confined geometries on long term health and stability of cell lines that are not strictly contact dependent. Orner et. al. [36] discussed hES cell attachment to laminin-derived peptides deposited in 750 μm squares, but the hES cells were only cultured for two days before cellular analysis. After two days, significant spontaneous generation is unlikely to occur even in suboptimal conditions, and no cell characterization data (e.g., differentiation or viability) was presented [32]. Although short-term analysis of selective attachment is useful for screening substrates that permit cell adherence and initial replication, several other requirements exist for use as a robust culture technique with hES cells. That is, hES cells must remain viable, undifferentiated, retain ability for undifferentiated proliferation upon passaging and remain pluripotent. Because hES cell differentiation does not occur immediately, short-term analysis may not accurately represent hES cell response to confinement.
Three-dimensional (3-D) microwells have also been used to study effects of confinement on short-term culture of anchorage-dependent cells. For example, NIH 3T3 fibroblasts were deposited as single cells in microwells 15 μm deep, 75 μm2 cross-sectional area [15]. These cells, however, were incubated for only four hours to investigate initial cell attachment and spreading, rather than long-term behavior in microwells. Single epithelial cells were also deposited in 11 μm deep×10 μm lateral microwells. Unfortunately, cell viability after two days was determined solely by visual cell replication [29]. These studies demonstrated the possibility of cell attachment in microwells, but did not show a marked improvement over prior patterned microwells that also constrained cells for at least two days.